Abstract

We investigated mitotic delay during replication arrest (the S-M checkpoint) in DT40 B-lymphoma cells deficient in the Chk1 or Chk2 kinase. We show here that cells lacking Chk1, but not those lacking Chk2, enter mitosis with incompletely replicated DNA when DNA synthesis is blocked, but only after an initial delay. This initial delay persists when S-M checkpoint failure is induced in Chk2-/- cells with the Chk1 inhibitor UCN-01, indicating that it does not depend on Chk1 or Chk2 activity. Surprisingly, dephosphorylation of tyrosine 15 did not accompany Cdc2 activation during premature entry to mitosis in Chk1-/- cells, although mitotic phosphorylation of cyclin B2 did occur. Previous studies have shown that Chk1 is required to stabilize stalled replication forks during replication arrest, and strikingly, premature mitosis occurs only in Chk1-deficient cells which have lost the capacity to synthesize DNA as a result of progressive replication fork inactivation. These results suggest that Chk1 maintains the S-M checkpoint indirectly by preserving the viability of replication structures and that it is the continued presence of such structures, rather than the activation of Chk1 per se, which delays mitosis until DNA replication is complete.

Checkpoint kinase expression in WT, Chk1−/−, and Chk2−/− cells and detection of mitotic cells by flow cytometry. (A) Western blot analysis of Chk1 (left) and Chk2 (right) expression in the cell lines used for this study. (B) Asynchronous cultures of WT, Chk1−/−, or Chk2−/− cells were separated by centrifugal elutriation to generate purified G1/S cell populations. A representative example of a WT cell culture before and after purification is shown. The purified G1/S populations were returned to culture in the presence or absence of 20 μM aphidicolin and the continuous presence of 1 μg of nocodazole/ml. The cells were harvested and analyzed for DNA content and pSer10H3 fluorescence (pH3 fluorescence) by flow cytometry as shown in the bottom right panel (WT cells were cultured with nocodazole alone for 7 h).

Wild-type DT40 cells sustain a prolonged S-M checkpoint delay when DNA synthesis is inhibited. (A) pSer10H3 fluorescence-DNA content flow cytometry analysis of elutriated WT G1/S cells cultured with 1 μg of nocodazole/ml with or without (control [con]) 20 μM aphidicolin for the indicated times. Open boxes indicate total pSer10H3-positive cells (pH3), and the open arrow indicates pH3-positive cells with 4N DNA content. (B) Quantitation of the data shown in panel A. (C) Western blot analysis of total and Ser345-phosphorylated Chk1 in elutriated WT cells cultured for the indicated times as described for panel A.

Chk1 is required to maintain the S-M checkpoint. (A) pSer10H3 fluorescence-DNA content flow cytometry analysis of elutriated Chk1−/− G1/S cells cultured with 1 μg of nocodazole/ml with or without (control [con]) 20 μM aphidicolin for the indicated times. Open boxes indicate total pSer10H3-positive cells (pH3). The open arrow indicates pSer10H3-positive cells with a 4N DNA content, and the solid arrow indicates pSer10H3-positive cells with a 2N DNA content. (B) Quantitation of the data shown in panel A. (C) More detailed kinetic analysis of the rates at which Chk1−/− cells accumulated in premature or natural mitosis in the presence or absence of aphidicolin, performed as described for panel A. (D) Mitotic spindles in pSer10H3-positive Chk1−/− cells. Elutriated G1/S WT or Chk1−/− cells were treated with aphidicolin for 12 h without nocodazole, fixed, and stained with antibodies against pSer10H3 (red) and α-tubulin (green).

Cdc2-associated histone H1 kinase activity, Cdc2 Y15 phosphorylation, and cyclin B2 expression in elutriated WT and Chk1−/− cells during replication arrest. (A) Cdc2-associated H1 kinase activity and Western blot analysis of Tyr15-phosphorylated Cdc2, total Cdc2, and cyclin B2 protein levels in elutriated G1/S WT cultures with 1 μg of nocodazole/ml and with or without (control) 20 μM aphidicolin for the indicated times. The prominent band in the anti-Cdc2 Western blot analysis of the immunoprecipitation-kinase assay corresponds to the light chain of the immunoprecipitating antibody (also shown in panel B). The samples were split in two: one half was analyzed for H1 kinase activity and the second half was used for Western blot analysis. The pY15 Cdc2, total Cdc2, and cyclin B2 results shown in the lower parts of panels A and B were derived from sequential reprobing of the same Western blots. (B) Cdc2-associated H1 kinase activity and Western blot analysis of Tyr15-phosphorylated Cdc2, total Cdc2, and cyclin B2 protein levels in elutriated G1/S Chk1−/− cultures treated as described for panel A. (C) Levels of Tyr15-phosphorylated Cdc2, total Cdc2, cyclin B2, and actin proteins in total (unsorted) and sorted pSer10H3-positive (mitotic) and -negative (nonmitotic) populations of Chk1−/− cells treated with 1 μg of nocodazole/ml and 20 μM aphidicolin for 12 h to induce premature mitosis (left) or with 1 μg of nocodazole/ml alone for 12 h to enrich for natural mitotic cells (right). (D) Cyclin B2 was immunoprecipitated from cultures of WT and Chk1−/− cells treated with 1 μg of nocodazole/ml and 20 μM aphidicolin for 12 h, and the coprecipitated Cdc2 protein was visualized by Western blotting. The positions of phosphorylated (asterisk) and nonphosphorylated Cdc2 protein are indicated.

Mitotic delay in the absence of Chk1 and Chk2 activity. (A) Elutriated G1/S WT cells were incubated with 1 μg of nocodazole/ml in the presence or absence of 20 μM aphidicolin, with or without 300 nM UCN-01, for the indicated times. At each time point, the percentage of pSer10H3-positive cells was determined by flow cytometry as described in the legends for Fig. and . Also included for comparison are data for elutriated Chk1−/− cells treated with 20 μM aphidicolin alone (shaded triangles). (B) Elutriated G1/S Chk2−/− cells treated as described for panel A.

Loss of viable replication structures precedes checkpoint failure in Chk1-deficient cells. (A and B) Elutriated G1/S WT and Chk1−/− cultures were treated with 20 μM aphidicolin and 1 μg of nocodazole/ml for the indicated times, washed free of drugs, and pulse-labeled with BrdU for 1 h, and the percentages of BrdU-labeled (A) and pSer10H3-positive cells (B) were then determined by flow cytometry. The data shown are means and standard errors for three independent experiments. (C) Elutriated G1/S Chk1−/− cells were treated with 20 μM aphidicolin and 1 μg of nocodazole/ml for 12 h, washed free of drugs, and pulse-labeled with CldU for 1 h. The pSer10H3-positive and -negative populations (top) were then separated by cell sorting and stained for CldU incorporation, and the percentage of cells capable of DNA replication in each population was determined by fluorescence microscopy. Two hundred nuclei were scored in each case. (D) Same data as for panels A and B, except that the cultures were treated with 20 μM aphidicolin and 1 μg of nocodazole/ml for 12 h with or without the continuous presence of 100 μM Roscovitine, washed, and pulse-labeled with BrdU for 1 h, and the percentages of BrdU-labeled and pSer10H3-positive cells were then determined.

Loss of nuclear PCNA staining precedes premature mitosis in Chk1−/− cells. (A) Elutriated G1/S WT and Chk1−/− cells were incubated with 20 μM aphidicolin and 1 μg of nocodazole/ml for the indicated times, washed free of drugs, incubated in fresh medium for 1 h, fixed, and stained with antibodies specific for pSer10H3 and PCNA. The percentages of cells exhibiting detectable staining for pSer10H3 and PCNA were determined by scoring 200 cells at each time point by fluorescence microscopy. (B) Representative fields of WT and Chk1−/− cells after 12 h of replication arrest. Essentially all of the WT cells exhibited strong nuclear staining for PCNA but were uniformly negative for pSer10H3, whereas Chk1−/− cells exhibited mutually exclusive staining for PCNA (green) and pSer10H3 (red). Individual examples are indicated by white arrows.